Collagen-based adhesives and sealants and methods of preparation and use thereof

ABSTRACT

Collagen-based compositions as adhesives and sealants for medical use and preparation thereof are described. Prior to polymerization, soluble or partially fibrillar collagen monomers in solution are chemically modified with an acylating agent, sulfonating agent or a combination of the foregoing. The collagen compositions prepared accordingly can be used as medical adhesives for bonding soft tissues or be made in to a sealant film for a variety of medical uses such as wound closures and tendon wraps for preventing adhesion formation following surgery.

FIELD AND BACKGROUND OF THE INVENTION

The ability to establish bonding between biological tissues has longbeen a goal of biomedical researchers. Attempts to provide desiredadhesion through mechanical bonding have proven to be neither convenientnor permanent (Buonocore, M., Adhesion in Biological Systems, R. S.Manly, ed., Academic Press, New York, 1970, Chap. 15). For this reason,much attention was devoted to developing synthetic polymers, e.g.,cyanoacrylates, as biomedical adhesives. These plastic materials,however, have been observed to induce inflammatory tissue reaction.Moreover, the ability of these materials to establish permanent bondingunder physiological conditions has yet to be fully realized.

The known toxicity associated with synthetic adhesives has led toinvestigations towards the development of biologically derived adhesivesas bonding materials. Among such adhesives, fibrin based glues havecommanded considerable attention. (See, e.g., Epstein, G. H. et al. Ann.Otol. Rhinol. Laryngol. 95: 40-45 (1986); Kram, H. B et al. Arch. Surg.119: 1309-1311 (1984); Scheele, J. et al. Surgery 95: 6-12 (January1984); and Siedentop, K. H. et al. Laryngoscooe 93: 1310-1313 (1983) forgeneral discussion of fibrin adhesives). Commercial fibrin tissueadhesives are derived from human plasma and hence pose potential healthrisks such as adverse immunogenic reactions and transmission ofinfectious agents, e.g., Hepatitis B virus. Moreover, the bond strengthimparted by such adhesives are relatively weak compared to collagenadhesives (see De Toledo, A. R. et al. Asso. for Res. in Vision andOphthalmology, Annual Meeting Abstract, Vol. 31, 317 (1990).Accordingly, there is a need for safe, effective biologically compatibletissue adhesives for biomedical applications.

Collagen, the major connective tissue protein in animals, possessesnumerous characteristics not seen in synthetic polymers. Characteristicsof collagen often cited include good compatibility with living tissue,promotion of cell growth, and absorption and assimilation ofimplantations (Shimizu, R. et al. Biomat. Med. Dev. Art. Org., 5(1):49-66 (1977)). Various applications of this material are being tested,for example, as dialysis membranes of artificial kidney (Sterzel, K. H.et al. Ameri. Soc. Artif. Int. Organs 17: 293 (1971)), artificial cornea(Rubin, A. L. et al. Nature 230: 120 (1971) and U.S. Pat. No.4,581,030), vitreous body (Dunn, M. et al. Amer. Soc. Artif. Int. Organs17: 421 (1971)), artificial skin and blood vessels (Krajicek, M. et al.J. Surg. Res. 4, 290 (1964)), as hemostatic agents (U.S. Pat. No.4,215,200), soft contact lens (U.S. Pat. Nos. 4,264,155; 4,264,493;4,349,470; 4,388,428; 4,452,925 and 4,650,616) and in surgery (Chvapil,M. et al. Int. Rev. Conn. Tiss. Res. 6: 1-61 (1973)). Natural collagenfibers, however, are basically insoluble in mature tissues because ofcovalent intermolecular crosslinks that convert collagen into aninfinite crosslinked network. Dispersal and solubilization of nativecollagen can be achieved by treatment with various proteolytic enzymeswhich disrupt the intermolecular bonds and removes immunogenicnon-helical end regions without affecting the basic, rigidtriple-helical structure which imparts the desired characteristics ofcollagen (see U.S. Pat. Nos. 3,934,852; 3,121,049; 3,131,130; 3,314,861;3,530,037; 3,949,073; 4,233,360 and 4,488,911 for general methods forpreparing purified soluble collagen). Subsequent purification of thesolubilized collagen can be accomplished by repeated precipitation athigh pH or ionic strength, washing and resolubilization. Introduction ofcovalent crosslinks into the purified soluble collagen is an importantaspect in stabilizing and restructuring the material for biomedical use.

Various methods and materials have been proposed for modifying collagento render it more suitable as biomedical adhesives. (See, e.g., DeToledo, A. R. et al. Asso. for Res. in Vision and Ophthalmology, AnnualMeeting Abstract, Vol. 31, 317 (1990); Lloyd et al., "Covalent Bondingof Collagen and Acrylic Polymers," American Chemical Society Symbosiumon Biomedical and Dental Applications of Polymers, Polymer Science andTechnology, Vol. 14, Plenum Press (Gebelein and Koblitz eds.), New York,1980, pp. 59-84; Shimizu et al., Biomat. Med. Dev. Art. Org., 5(1):49-66 (1977); and Shimizu et al., Biomat. Med. Dev. Art. Org., 6(4):375-391 (1978), for general discussion on collagen and syntheticpolymers.) In many instances, the prior modified collagen-basedadhesives suffer from various deficiencies which include (1)crosslinking/polymerization reactions that generate exothermic heat, (2)long reaction times, and (3) reactions that are inoperative in thepresence of oxygen and physiological pH ranges (Lee M. L. et al.Adhesion in Biological Systems, R. S. Manly, ed., Academic Press, NewYork, 1970, Chap. 17). Moreover, many of the prior modifiedcollagen-based adhesives contain toxic materials, hence rendering itunsuitable for biomedical use (see, for example, Buonocore, M. G. (1970)and U.S. Pat. No. 3,453,222).

To date, there are no safe, efficacious adhesives for medical use withsoft tissue. Collagen-based adhesives with appropriate adhesive strengthwould have enormous utility in many medical applications, particularlyinvolving soft tissues. Such adhesives could be used to seal incisionsfollowing cataract removal and to attach epikeratophakic grafts tocorneal tissue, etc. Marketing research has indicated that there areover 8 million surgical procedures that could use a safe, effectivebiological adhesive.

SUMMARY OF THE INVENTION

The present inventors have discovered that a biologically compatible,collagenous reaction product with sealant and adhesive properties can beformed using chemically modified collagen. Modification of pure, solubleor partially fibrillar collagen monomers with an acylating agent or asulfonating agent or a combination of the foregoing, renders collagenmonomers soluble at physiological conditions. Subsequent polymerizationof the chemically modified monomers produces a polymerized collagencomposition with adhesive and sealant properties. The polymerizationreaction may be initiated with an appropriate polymerizion initiatorsuch as a chemical oxidant, ultraviolet irradiation, a suitableoxidative enzyme or atmospheric oxygen.

Accordingly, it is an object of the present invention to providepolymerized chemically modified collagen compositions as a safe,effective biological adhesives with appropriate adhesive strength forbiomedical applications, particularly involving soft tissues. Suchadhesives may be used to seal incisions following cataract removal andto attach epikeratophakic grafts to corneal tissue for correction ofrefractive errors.

It is another object of the present invention to provide a method todramatically improve the biostability of collagen formulations asdetermined by resistance to neutral proteases and to vertebratecollagenase.

It is still another object of the present invention to provide a numberof collagenous reaction products, that is, a polymerized biologicallycompatible collagenous reaction product useful in biomedicalapplications as adhesives and sealants of soft tissue. The polymerizedmaterials may assume a number of sizes and shapes consistent with theirintended biomedical applications, which include use in ophthalmology,plastic surgery, orthopedics and cardiology.

It is a further objective of the present invention to provide a processfor derivatization of chemically modified collagen monomers withacrylamide which is then polymerized to produce apolyacrylamide-collagen co-polymer. Such materials may be useful forproducing mechanical type adhesives, polymerization occurring after thesolutions penetrate tissue.

These and other objects will become more apparent in light of thedetailed description which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1a-c illustrates the attachment of a lenticule by application of acollagen-based sealant at the periphery of the lenticule inepikeratoplastic procedures.

FIGS. 2a-c illustrates the attachment of a lenticule by application of acollagen-based sealant on top of the lenticule in epikeratoplasticprocedures. The applied collagen-based sealant coats and seals thelenticule in its entirety.

DETAILED DESCRIPTION OF THE INVENTION

As employed herein, the term "biologically compatible" refers tocollagen modified in accordance with the present invention (i.e., apolymerized collagenous reaction product) which is incorporated orimplanted into or placed adjacent to the biological tissue of a subjectand more particularly, does not deteriorate appreciably over time orinduce an immune response or deleterious tissue reaction after suchincorporation or implantation or placement.

The type of collagen useful to form the biologically compatiblecollagenous reaction product with adhesive properties of this inventionis selected from the following groups: purified Type I collagen, Type IVcollagen and Type III collagen, intact collagen-rich tissue or acombination of any of the foregoing. Preferred as a collagen startingmaterial is purified Type I collagen. Type I collagen is ubiquitous andreadily extracted from animal tissues such as dermis and tendon. Commonsources are bovine tendon and hide and rat tail tendon. Extraction fromhuman tissues is difficult. U.S. Pat. No. 4,969,912, "Human CollagenProcessing and Autoimplant Use", describes unique methods to disperseand solubilize human tissue.

A variety of collagen solubilization procedures that are well known inthe art can be used to prepare soluble collagen solutions useful for theinstant invention. Native collagen is liberated from non-collagenconnective tissue constituents (lipids, sugars, proteins, etc.) andisolated after subjecting it to proteolytic enzymatic treatment by anenzyme other than collagenase. Suitable proteolytic enzymes includepronase and pepsin. The enzymatic treatment removes the immunogenicnon-helical portions of native collagen (telopeptide) and provides amonomeric collagen material which is soluble in dilute acidic aqueousmedia. A solution containing crude solubilized collagen is thensubjected to a series of treatments to purify the soluble atelopeptidecollagen from insoluble collagen, protease and non-collagen productsresulting from the proteolytic enzymatic procedure. Conventional methodsfor preparing pure, acid soluble, monomeric collagen solutions bydispersing and solubilizing native collagen are described, for example,in U.S. Pat. Nos. 3,934,852; 3,121,049; 3,131,130; 3,314,861; 3,530,037;3,949,073; 4,233,360 and 4,488,911. A method for preparing a collagensolution is provided in the examples that follow.

Suitable acylating agents for use in the instant invention includealiphatic, alicyclic and aromatic anhydrides or acid halides.Non-limiting examples of acylating agents include glutaric anhydride,succinic anhydride, lauric anhydride, diglycolic anhydride,methylsuccinic anhydride, methyl glutaric anhydride, dimethyl glutaricanhydride, succinyl chloride, glutaryl chloride, and lauryl chloride.These chemicals are available from Aldrich Chemical Company (Milwaukee,Wis). Preferred acylating agent for use in the present invention isglutaryl anhydride. An effective amount of an acylating agent is broadlyfrom about 0.5 to 7.5% wt total collagen, preferably from about 1.5 to5.0% total collagen in solution.

In addition, acylating agents having secondary reactive functionalitieswithin their chemical structure are also useful for modifying collagenmonomers. Examples of secondary functionalities include by way ofnon-limiting examples: epoxy, cyano, halo, alkenyl, and alkynyl.Non-limiting examples of acylating agents bearing secondaryfunctionalities include exo-3,6-epoxy-1,2,3,4-tetrahydrophthalicanhydride, methacrylic anhydride, 3,6-endoxo-3-methylhexahydrophthalicanhydride, and endo-3,6-dimethyl-3,6-endoxohexa hydrophthalic anhydride.Preferred as such acylating agents areexo-3,6-epoxy-1,2,3,4-tetrahydrophthalic anhydride and methacrylicanhydride. Without being bound by theory, the secondary functionalitiespresent in acylating agents can react covalently with amino acidresidues under acylation conditions or during polymerization.

Useful sulfonating agents for the preparation of modified collagenmonomers of the present invention include aliphatic, alicyclic andaromatic sulfonic acids or sulfonyl halides. Non-limiting examples ofsulfonating agents for use in the present invention includeanthraquinone-1, 5-disulfonic acid, 2-(chlorosulfonyl)-anthraquinone,8-hydroxyquinoline sulfonic acid, 2-naphthalene- sulfonyl chloride,beta-styrene sulfonyl chloride and 2-acrylamido-2-methyl-1-propanesulfonic acid. These chemicals are available from Aldrich ChemicalCompany (Milwaukee, Wis.). Preferred sulfonating agents for preparingthe adhesive collagen materials are anthraquinone-1, 5-sulfonic acid and2-(chlorosulfonyl)-anthraquinone. Such compounds, in non-toxic effectiveamounts, can be safely employed in collagen-based compounds for medicaluse as adhesives and sealants. An effective amount of sulfonating agentis broadly from about 0.5 to 20% wt total collagen, preferably fromabout 1.5 to 7.3 wt% total collagen in solution.

When a combination of an sulfonating agent and acylating agent is usedfor preparation of modified collagen monomers, the amount of acylatingagent and sulfonating agent in total, is preferably from about 1.5 to7.5% wt of collagen in solution. Excess quantities of chemical modifiersbeyond the preferred range may result in a collagen composition that isbiologically unstable and sensitive to tissue proteases.

Acylation of collagen is carried out at alkaline pH, for example, in therange from about 7.5 to 10.0, preferably from about 8.5 to 9.5, and morepreferably at about pH 9.0. The acylation reaction can be monitored bythe decrease in pH. The reaction is complete when pH remains stable. Thereaction can also be monitored by removing aliquots and measuring thefree amine concentration of precipitated, washed collagen product.

The acylation reaction should be complete in about 5 to about 90minutes, preferably from about 20 to 40 minutes. The reactions should becarried out at temperatures from about 4° C. to 37° C., preferably fromabout 4° C. to 25° C.

The reaction can be stopped by adjusting the pH to 12.0 for 2 minutes.This destroys residual, unreacted acylating agent. The modified collagenis then precipitated by reducing the pH using hydrochloric acid, aceticacid, nitric acid, sulfuric acid, or other acid.

The amount of acid must be sufficient to precipitate out the chemicallyderivatized collagen. Generally precipitation occurs at a pH of betweenabout 3.5 and 6.0, preferably between about 4.0 and 5.0.

The precipitate of reacted collagen which now contains substituentgroups reacted with amine groups (primarily epsilon-amino groups), isrecovered from the mixture using conventional techniques such ascentrifugation or filtration. Centrifugation at about 3,000 to about15,000 rpm for about 20 to 60 minutes, preferably from about 4,000 to12,000 for about 20 to 30 minutes provides efficient recovery of theprecipitate.

After recovery, the precipitate is washed with deionized water andsubsequently dissolved in a physiological solution, e.g., phosphatebuffer (0.1M) at pH 7.2. It is often necessary to adjust the pH fromabout 7.0 to 7.5 by addition of sodium hydroxide solution.

Following dissolution of the precipitate, the solution is generallyfiltered by conventional filtering means, i.e. a 5 micron filter, andthen centrifuged to remove air bubbles. At this point, the resultingsolution containing chemically modified collagen monomers exhibits aviscous consistency and varying degrees of transparency and claritydepending on the extent of acylation and on the state of solubility ofthe starting collagen material.

The extent of acylation determines the biological stability of theresultant sealant or adhesive structure. Complete acylation, reactionwith all available free amines, produces a collagen composition withmaximum amounts of adhesive moieties. However, such a material may notbe biologically stable. Complete acylation results in a collagen sealantthat rapidly degrades in the presence of neutral proteolytic enzymes,such as trypsin. It has been discovered that the biological stability ofresultant sealants and adhesive systems can be manipulated bycontrolling the extent of acylation. The extent of acylation may bemodulated by varying the amount of acylation agent, the pH, thetemperature and the time of the reaction. In addition, the method ofaddition of the acylating agents will affect the reaction. Reactions aregenerally slower if the acylating agent is added as a solid or powderrather than as a solution.

Biological stability of the polymerized collagen compositions appears tobe additionally affected by the solubility characteristics of thestarting collagen. Completely soluble collagen generally does notproduce a sealant or adhesive that is resistant to neutral proteolyticenzymes. If the starting collagen solution is adjusted to pH 7.0-7.6 andallowed to undergo limited fibrillogenesis at 25° to 37° C. beforechemical modification, the final sealant or adhesive complex isresistant to degradation by neutral proteolytic enzymes, such astrypsin.

The chemically modified collagen solution thus obtained is subsequentlysubjected to polymerization or crosslinking conditions to produce thepolymerized collagen composition of the present invention.Polymerization may be carried out using irradiation, e.g., UV, gamma, orfluorescent light. UV irradiation may be accomplished in the short wavelength range using a standard 254 nm source or using UV laser sources.With a standard 254 nm source, 4-12 watts, polymerization occurs from 10to 40 minutes, preferably 20 to 30 minutes, at an exposure distance offrom 2.5-10 cm, preferably from 2.5 to 5 cm distance. Excess UV exposurewill begin to depolymerize the collagen polymers. Polymerization usinggamma irradiation can be done using from 0.5 to 2.5 Mrads. Excess Gammaexposure will also depolymerize collage polymers. Polymerization in thepresence of oxygen can be done by adding an initiator to the fluid priorto exposure. Non-limiting examples of initiators include sodiumpersulfate, sodium thiosulfate, ferrous chloride tetrahydrate, sodiumbisulfite and oxidative enzymes such as peroxidase or catechol oxidase.When initiators are employed, polymerization occurs in 30 seconds to 5minutes, usually from 1 to 3 minutes.

Preferred as a polymerizing agent is UV irradiation. However, thepolymerization or crosslinking of the monomeric substituents can becarried out by simply exposing the material to atmospheric oxygen,although the rate of polymerization is appreciably slower than in thecase of UV irradiation or chemical agents.

In one embodiment of the present invention, collagen products containing2-acrylamido-2-methyl-1-propane sulfonic acid may be mixed with N,N-methylene bisacrylamide to produce mechanical sealants and adhesivesystems. Addition of initiators such as sodium persulfate or ammoniumpersulfate, and exposure to fluorescent light rapidly results inpolymerization to form polyacrylamide-collagen co-polymers. Collagenproducts containing methacrylic acid polymerize spontaneously in air.This polymerization can be accelerated under UV irradiation in theabsence of oxygen.

In another embodiment of the present invention, the polymerizedcollagenous reaction products can be made in the form of a sealant film.As described in the examples which follow, such a film is flexible andelastic with the consistency and feel of plastic film, and yet the filmexhibits high biological compatibility. Uses of sealant films include:Prevention of adhesion formation following tendon surgery (i.e., use asa wrap around tendons), use as a synthetic tympanic membrane, substitutefacial tissue and wound dressing component. Additional examples ofpotential usage of sealant films include: treatment of cornealabrasions, wound closure, coating of catheters and instruments, use as amaterial to prevent adhesion formation in tissues other than tendons(e.g., peritoneal cavity).

Also as further embodiments of the present invention, the sealant andadhesive formulations can be used as systems specific for delivery ofnumerous drugs, growth factors, and biological compounds. Such materialscan be added to the collagen adhesive or sealant to promote cellmigration, cell adhesion, and wound healing.

The working examples set forth below are intended illustrate theinvention without limiting its scope.

EXAMPLE 1 A. Preparation of Acid Soluble Type I Collagen Solution

Fibrous Type I collagen was prepared from bovine material (calf hide)using the following procedure:

Clean, dehaired split hides were purchased from the Andre ManufacturingCo. (Newark, N.J.) and frozen until ready for use. Approximately 200 gof calf hide was thawed at room temperature and cut into approximately 1cm³ pieces using a scalpel and tweezers. The weight of the wet tissuewas recorded. The calf hide was then placed into 15 liters of 0.5Macetic acid and stirred with a lightening mixer at room temperature forat least one hour. A ten mL solution of 0.5M acetic acid containing 2%w/w (or 3.9 g) pepsin from porcine mucosa (Sigma Chemicals, St. Louis,Mo.) was added to the calf hide solution. This solution was stirredovernight with a lightening mixer at room temperature. An additional tenmL 0.5M acetic acid solution containing 1% w/w (or 1.96 g) pepsin wasadded to the calf hide mixture. The solution was again stirred overnightwith a lightening mixer at room temperature. The dissolved calf hidesolution was refrigerated until a uniform temperature of 4° C. wasreached, a process that may take overnight. The pH of the solution wasadjusted to 9.0 with 10 N NaOH to denature pepsin. Stirring wasmaintained throughout the pH adjustment process with a lightening mixer.As collagen will precipitate out at pH 9.0 when the temperature is above6° C., ice cubes may be added directly to maintain the 4° C temperature.The solution is then refrigerated for at least four hours, thencentrifuged at 4° C. for 30 minutes at 9 rpm. The resulting pellet,containing pepsin, was discarded. The supernatant, containing collagen,was subjected to a series of purification steps.

Collagen was precipitated out by addition of solid NaCl to thesupernatant to a final concentration of 2.5M. The solution was stirredat room temperature for at least two hours. The collagen precipitate wascollected by centrifugation of solution for 30 minutes at 9000 rpm andredissolved in 15 liters of 0.5M acetic acid, a process requiring atleast 2 hours. Collagen was reprecipitated out again by addition ofsolid NaCl to the solution to a final concentration of 0.8M. Thesolution stirred for at least two hours and the collagen was collectedby centrifugation of the solution for 30 minutes at 9000 rpm. Thisredissolving/precipitation procedure was repeated once more. The finalpellet, containing purified collagen, was dissolved in 0.1M acetic acidof sufficient volume to provide approximately 0.3% w/w collagen Type Isolution of pH 3.0. The collagen solution was then prefiltered through a0.3 micron filter and sterilized through a 0.22 micron filter. Thecollagen solution can now be used in the modification process.

B. Preparation of Anthraquinone Collagen Using2-(Chlorosulfonyl)-Anthraquinone

Pure collagen was prepared as previously described. Following filtrationthrough a 0.2 micron filter, 100 ml of collagen solution was brought topH 9.0 and reacted with 6.0 mg of 2-(chlorosulfonyl)-anthraquinone andthen 6.0 mg of glutaric anhydride. These chemicals were added in solidform while maintaining the pH at 9.0. After 3 hours, the pH was reducedto 4.5 to precipitate the modified collagen. The precipitate was washedthree times with deionized water and subsequently dissolved in 0.005Mphosphate buffer, pH 7.5, containing 2% glycerol. The pH was adjusted to7.4 using sodium hydroxide. The material was slightly cloudy and wasfiltered through a 5 micron filter. Approximately 50 ml of 20 mg/mlsodium persulfate was added to 5 ml of the collagen and the mixtureexposed to 254 nm UV irradiation for 30 seconds. A firm, flexible filmwas formed. The same material before UV exposure was used to attach twopieces of bovine dermis. Two sections of dermis approximately 1×1 cmwere placed in a glass petri dish side by side. Collagen fluidcontaining sodium persulfate was painted on each dermis section and overthe space adjoining the dermis sections. The sections were then exposedto 254 nm UV irradiation at about 3 cm distance from the source. Afterexposure for about 60 seconds, the dermis sections were joined. Thestrength of the joint was not measured. However, the dermis sectionsremained joined after incubation in sterile 0.9% sodium chloridesolution for several weeks.

In another experiment, anthraquinone collagen solution alone (withoutpersulfate) was used to join two sections (each 1×1 cm) of dermaltissue. The sections were exposed to 254 nm UV irradiation for twominutes. The dermal sections became joined and were placed in sterile0.9% sodium chloride solution. The sections remained attached even afterseveral weeks.

C. Preparation of Anthraquinone Collagen Using Anthraquinone-1,5-Disulfonic Acid

Pure, acid soluble collagen was prepared as previously described.Following filtration through a 0.2 um filter, 100 ml of collagensolution was brought to pH 9.0 and reacted with 6.0 mg ofanthraquinone-1, 5-disulfonic acid and 6 mg of glutaric anhydride. Thesechemicals were added in solid form while maintaining the pH at 9.0.After 30 minutes, the pH was reduced to 4.0 to precipitate the modifiedcollagen. The precipitate was washed three times with sterile water andsubsequently dissolved in 0.005M phosphate buffer containing 2%glycerol. The pH was adjusted to 7.4 using 1N NaOH. The material wasslightly cloudy. A 50 ul aliquot of sodium persulfate (20 mg/ml) wasadded to the modified collagen and the material exposed to UVirradiation for 30 seconds. A firm, but flexible mass resulted. The samematerial before UV irradiation was used to attach two sections of bovinecorium. Following brief UV irradiation (30 sec.), the sections becamebound together. The tear strength was not measured but appearedsufficient to resist substantial force.

EXAMPLE 2: SEALING SYNTHETIC LENTICULES IN EPIKERATOPHAKIA

In this example, a 10 ul aliquot of sodium persulfate (20 mg/ml) wasadded to a solution containing collagen modified with glutaric anhydridealone. The mixture was placed on the surface of an intact, enucleatedbovine eye, and allowed to flow over the surface of the eye for about 2minutes. At that point, the eye was exposed to UV irradiation for 30seconds. The collagen mixture polymerized into a thin, somewhat flexiblefilm that covered the eye surface.

In another experiment, glutaric modified collagen without sodiumpersulfate was added to the surface of an intact, enucleated bovine eye.The eye was then placed in an atmospheric chamber, the chamber flushedwith nitrogen, and the eye with collagen coating exposed to UVirradiation for 20 minutes. Again, there appeared to be a smooth, veryflexible film of collagen covering the eye.

Such a sealant could be useful for attaching a synthetic or naturallenticule in epikeratoplastic procedures. In this case the synthetic ornatural lenticule would be positioned on the corneal surface. Thecollagen sealant would then be placed over the lenticule and adjacentcorneal tissue and polymerized in place to seal the lenticule to theadjacent corneal tissue. For example, the collagen-based sealant can beapplied on the periphery of the lenticule and adjacent corneal tissue(FIG. 1a), then exposed to UV irradiation (FIG. 1b) to seal thelenticule in place (FIG. 1c). Alternatively, the collagen-based sealantcan be placed on top of lenticule (FIG. 2a) which then flows over andcoats the surface of the lenticule and adjacent corneal tissue. Thecoating is then exposed to UV irradiation (FIG. 2b) which seals thelenticule in place (FIG. 2c). Polymerization using UV irradiation mustbe accomplished in the absence of oxygen. Thus, formulations containinga safe initiator are more practical, eliminating the need to excludeoxygen.

EXAMPLE 3: RESISTANCE TO NEUTRAL PROTEASE AND COLLAGENASE AND USE FORLENTICULE ATTACHMENT

In this example, pure, soluble collagen was prepared as previouslydescribed. Following filtration through a 0.2 um filter, the solutionwas brought to pH 7.0 and placed in a water bath at 37° C. to initiatecollagen fibrillogenesis. After approximately 15 minutes, the solutionbecame cloudy. At this point 100 ml of solution at 2.8 mg/ml collagenwas adjusted to pH 9.0 and 7.0 mg of glutaric anhydride was added. Thereaction was allowed to continue for another 20 minutes. The pH was thendecreased to 4.3 to precipitate the modified collagen. The precipitatewas recovered by centrifugation and was washed three times. The finalprecipitate was very fine and granular. This was dissolved in phosphatebuffer and adjusted to pH 7.0-7.4. The viscous solution was clear toslightly cloudy. Films were prepared and exposed to UV irradiation for20 minutes. Samples were then evaluated for resistance to neutralprotease (trypsin) and to vertebrate collagenase. These results werecompared to standard glutaric collagen (excess glutaric anhydride).Table 1 shows the results from such evaluation.

                  TABLE 1                                                         ______________________________________                                        RESISTANCE TO NEUTRAL PROTEASE AND                                            COLLAGENASE RESISTANCE (%), 24-25 HOURS                                       Buffer        Trypsin  Vertebrate Collagenase                                 ______________________________________                                        Standard                                                                              95        30       30                                                 Glutaric                                                                      Resistant                                                                            100        87.4     94.7                                               Glutaric                                                                      ______________________________________                                    

The standary glutaric formulation was examined in situ by placing asample of the material on the surface of a lenticule and then exposingthe material to UV irradiation (short wave length) for 20 minutes in anitrogen atmosphere. The collagen material was formed into a thin solidfilm which covered the entire lenticule and seemed to provide acontinuous tapering attachment to Bowman's membrane. Epithelial cellmigration and attachment had started over the collagen film. However,some of the collagen film began to disintegrate before epithelial cellmigration was complete. This probably was caused by epithelial cellproteases.

EXAMPLE 4: ADHESIVE FORMULATION

Glutaric modified collagen was prepared using the procedures describedin Example 3. The modified collagen was redissolved at about 5%concentration in phosphate buffer (0.005M at pH 7.5) containing 2%glycerol. This high concentration material was supplemented with sodiumpersulfate (100 ul of a 20 mg/ml solution) per 2 ml of redissolvedcollagen and used to join two sections of light cardboard. The fluid waspainted onto the surfaces of the adjoining cardboard and allowed to wetthe surfaces. The pieces were then exposed to UV irradiation for 2minutes. After exposure, a crude measure of bond strength was made byattaching one piece of cardboard to a clamp suspended from a ring standand attaching weights to the other piece of cardboard. The joint did notbreak after adding up to 50 grams of weight.

EXAMPLE 5: EPOXY-COLLAGEN ADHESIVE

A collagen solution was obtained as previously described. Afterfiltration through a 0.2 filter, 300 ml of collagen solution was broughtto pH 7.5 and placed in a water bath at 37° C. until fibril formationwas initiated. The pH of the solution was then raised to 9.0 and 42 mgof exo-3,6-epoxy-1,2,3,4-tetrahydrophthalic anhydride added in 10 dropsof dimethyl formamide. The pH was maintained at 9.0 for 30 minutes.After 30 minutes the pH was dropped to 7.5 and the solution allowed toequilibrate for an additional 30 minutes. The neutral solution wascentrifuged at 9,000 rpm for 20 minutes, the supernatant removed and pHadjusted to 4.3 to precipitate the neutral soluble, epoxy-modifiedcollagen. The precipitate was washed three times with sterile water andreconstituted in 0.005M phosphate buffer, pH 8.6, containing 2% sterileglycerol. The resulting solution was clear, transparent and viscous. Onealiquot of about 5 ml was removed and exposed to 254 nm UV light for 5minutes. The material thickened but did not polymerize to a hard film.Another aliquot of 5 ml was removed and 25 ul of sodium persulfate (20mg/ml) added. This was also exposed to 254 nm UV irradiation for 2minutes. The material polymerized to a relatively firm, but flexiblefilm.

The modified collagen was then used to attach two sections of porouspaper. The bond was strong and required significant force to separatethe two sections. This same material was used to attach two sections ofcalf skin. UV irradiation in the absence of oxygen, was used as theinitiator in place of the sodium persulfate. After 20 minutes, thesections were firmly attached.

In still another case, an aliquot of epoxy-collagen was added to twosections of porous paper and placed in the laminar flow hood for 10minutes. The material polymerized and dried to a firm and extremelyflexible film that bonded the two sections of paper.

EXAMPLE 6: METHACRYLIC COLLAGEN ADHESIVE AND TESTING

In this example, 200 ml of soluble collagen was modified at pH 9.0 with30 mg of methacrylic anhydride. The reaction continued for 30 minutesafter which 10 mg of glutaric anhydride was added and reacted foranother 30 minutes. The modified collagen was precipitated by adjustingthe pH to 4.5. The precipitate was recovered by centrifugation andwashed three times with sterile water. The material was reconstituted in0.005M phosphate buffer containing 2% glycerol. The solution was clearand viscous. An aliquot was removed and exposed to 254 nm UVirradiation. After 2 minutes, the material had formed a clear, firmfilm. Another aliquot was removed and placed on a microscope slide.After 30 minutes, the material had polymerized spontaneously to form aclear, firm film. Two sections of alcohol washed calf skin were placedon a microscope slide, and the interface coated with methacryliccollagen. After 16 hours, the sections were firmly attached. This samplewas then incubated in sterile buffer at room temperature. After oneweek, the sections remained firmly attached.

EXAMPLE 7: HUMAN TISSUE ADHESIVE MATERIAL

In this example, human dermis was dissected from full thickness skinspecimen and blended using an OMNI homogenizer with a Macro generator(10 mm). The tissue did not pulverize in buffer or saline. To the tissuewas added 5 mg of methacrylic anhydride per 200 mg of tissue. The tissueimmediately pulverized to a fine suspension. A second aliquot ofmethacrylic anhydride was added to further solubilize the tissue. Thepulverized tissue was centrifuged to separate the soluble fraction fromthe dispersed fraction. The modified tissue in the soluble fraction wasrecovered by adding 3 volumes of 70% ethanol. The collagen immediatelyformed fibers. These were recovered by centrifugation and dried in alaminar flow hood. The dried material was then reconstituted in 0.005Mphosphate buffer, pH 8.5, containing 2% glycerol. The thick solution wasslightly cloudy and viscous. This chemically modified tissue(methacrylic) was exposed to 254 nm UV light for 2 minutes to form astrong, flexible film which could potentially be used as an adhesive tobond tissue.

In another experiment, human tissue was treated with glutaric anhydrideinstead of methacrylic anhydride. Two aliquots of glutaric anhydride (15mg/200 mg tissue) were used to disperse and solubilize the human tissue.The soluble fraction was isolated and reconstituted in buffer, as above.The viscous material was extremely sticky. One aliquot was placed on amicroscope slide cover slip. After drying the droplets could not beremoved from the cover slip, even with a razor blade.

What is claimed is:
 1. A collagen composition comprising a polymerizableproduct formed by a reaction between (a) a partially fibrillar collagenderived by subjecting an acid solubilized collagen to a pH ranging from7.0 to 7.6 and a temperature ranging from 25° C. to 37° C. for a timesufficient to initiate fibrillogenesis and (b) at least one of anacylating agent and sulfonating agent.
 2. The collagen composition ofclaim 1 wherein the composition is biologically compatible.
 3. Thecollagen composition of claim 1 wherein the collagen is a purified TypeI collagen, purified Type III collagen, purified Type IV collagen,collagen rich tissue or a combination of the foregoing.
 4. The collagencomposition of claim 3 wherein the Type I collagen is derived from humantissue or animal tissue.
 5. The collagen composition of claim 4 whereinthe Type I collagen comprises autogenic human tissue.
 6. The collagencomposition of claim 1 wherein the acylating agent is glutaricanhydride, succinic anhydride, lauric anhydride, diglycolic anhydride,methyl succinic anhydride, methyl glutaric anhydride, dimethyl glutaricanhydride, exo-3,6-epoxy-1,2,3,4-tetrahydrophthalic anhydride,3,6-endoxo-3-methyl hexahydrophthalic anhydride,endo-3,6-dimethyl-3,6-endoxohexahydrophthalic anhydride, methacrylicanhydride, succinyl chloride, glutaryl chloride, lauryl chloride, or acombination of the foregoing.
 7. The collagen composition of claim 6wherein the acylating agent is glutaric anhydride.
 8. The collagencomposition of claim 6 wherein the acylating agent is methacrylicanhydride.
 9. The collagen composition of claim 6 wherein the acylatingagent is exo-3,6-epoxy-1,2,3,4- tetrahydrophthalic anhydride.
 10. Thecollagen composition of clam 1 wherein the sulfonating agent isanthraquinone-1, 5-disulfonic acid, 2-(chlorosulfonyl)-anthraquinone,2-acrylamido-2-methyl-1-propane sulfonic acid,8-hydroxyquinoline-5-sulfonic acid, beta-styrene sulfonyl chloride or acombination of the foregoing.
 11. The collagen composition of claim 10wherein the sulfonating agent is 2- (chlorosulfonyl)-anthraquinone oranthraquinone-1, 5-disulfonic acid.
 12. The collagen composition ofclaim 1 wherein the composition is polymerizable by exposing to UV andan initiator selected from the group comprising sodium persulfate,sodium thiosulfate, ferrous chloride tetrahydrate, sodium bisulfite or acombination of the foregoing.
 13. A collagen composition comprising apolymerizable product formed by a reaction between (a) a partiallyfibrillar collagen derived by subjecting an acid solubilized collagen toa pH ranging from 7.0 to 7.6 and a temperature ranging from 25° C. to37° C. for a time sufficient to initiate fibrillogenesis; (b) glutaricanhydride; and (c) a sulfonating agent.
 14. The collagen composition ofclaim 13 wherein the sulfonating agent is anthraquinone-1, 5-disulfonicacid, 2-(chlorosulfonyl)-anthraquinone, 2-acrylamido-2-methyl-1-propanesulfonic acid, 8-hydroxyquinoline-5-sulfonic acid, beta-styrene sulfonylchloride or a combination of the foregoing.
 15. The collagen compositionof claim 14 wherein the sulfonating agent is2-(chlorosulfonyl)-anthraquinone or anthraquinone-1, 5-disulfonic acid.16. A collagen composition comprising a polymerizable product formed bya reaction between (a) a partially fibrillar collagen derived bysubjecting an acid solubilized collagen to a pH ranging from 7.0 to 7.6and a temperature ranging from 25° C. to 37° C. for a time sufficient toinitiate fibrillogenesis; and (b) glutaric anhydride.
 17. A method formaking a collagen composition comprising the steps of:(a) preparing apartially fibrillar collagen by subjecting an acid soluble collagen to apH ranging from 7.0 to 7.6 and a temperature ranging from 25° C. to 37°C. for a time sufficient to initiate fibrillogenesis; and (b) reactingsaid partially fibrillar collagen with at least one of an acylatingagent and sulfonating agent at a pH ranging from 7.5 to 10.0 and at atemperature ranging from 4° C. to 37° C.
 18. The method of claim 17wherein step (b) is carrier out at a pH ranging from about 8.5 to 9.5.19. The method of claim 17 wherein step (b) is carried out at atemperature ranging from about 4° C. to 25° C.
 20. The method of claim17 wherein step (b) is carried out for a time period ranging from about5 to about 90 minutes.
 21. The method of claim 17 wherein at the leastone of an acylating agent and sulfonating agent is present at aconcentration between about 0.5 to 20 wt. % total collagen.
 22. Themethod of claim 21 wherein the concentration ranges between about 1.5 to7.5 wt. % total collagen.
 23. The method of claim 17 wherein theacylating agent is glutaric anhydride, succinic anhydride, lauricanhydride, diglycolic anhydride, methyl succinic anhydride, methylglutaric anhydride, dimethyl glutaric anhydride,exo-3,6-epoxy-1,2,3,4-tetrahydrophthalic anhydride, 3,6-endoxo-3-methylhexahydrophthalic anhydride,endo-3,6-dimethyl-3,6-endoxohexahydrophthalic anhydride, methacrylicanhydride, succinyl chloride, glutaryl chloride, lauryl chloride, or acombination of the foregoing.
 24. The method of claim 23 wherein theacylating agent is glutaric anhydride.
 25. The collagen composition ofclaim 23 wherein the acylating agent is methacrylic anhydride.
 26. Thecollagen composition of claim 23 wherein the acylating agent isexo-3,6-epoxy-1,2,3,4-tetrahydrophthalic anhydride.
 27. The method ofclaim 17 wherein the sulfonating agent is anthraquinone-1, 5-disulfonicacid, 2-(chlorosulfonyl)-anthraquinone, 2-acrylamido-2-methyl-1-propanesulfonic acid, beta-styrene sulfonyl chloride and8-hydroxyquinoline-5-sulfonic acid or a combination thereof.
 28. Themethod of claim 27 wherein the sulfonating agent is2-(chlorosulfonyl)-anthraquinone or anthraquinone 1, 5-disulfonic acid.